With the well-developed DNA recombinant technology, more and more proteins are being developed as pharmaceuticals. Development of stable protein formulations is one of the critical steps in developing protein as a therapeutic product. A vast amount of research has been conducted regarding protein stability, and this information is readily available in the literature. (a. Pearlman, R. and Wang, Y. J., 1996. Formulation, characterization, and stability of protein drugs. Plenum Press, New York. B. Chang, B. S. and Hershenson S., Chapter 1, Practical Approaches to Protein Formulation Development, Rational Design of Stable Protein Formulations, edited by Carpenter and Manning Kluwer Academic & Plenum Publishers, New York, 2002) However, due to the very nature of proteins, it is not practical for most of proteins to have only the native form of a protein in the formulation. More or less, proteins are formulated into solutions or solids (lyophilized, spray-dried, spray-freeze-dried) together with excipients to maximize protein stability during manufacturing process and during storage.
Besides various chemical reactions, such as disulfide scrambling, deamidation, peptide cleavage, oxidation, a major instability problem relates to non-covalent aggregation that is often immunogenic and sometimes produces precipitates. In liquid formulation, optimization of pH, ionic additives, aminoacid, surfactants, protein concentration and raw material purity may provide solutions to the aggregation problem. In solid formulation, proteins are usually formulated together with some buffer reagents, salt, and some bulky reagents such as mannitol, sucrose, trehalose, citric acid etc. to physically separate protein molecules, and thus to reduce aggregate formation.
Chaperones can also reportedly reduce or prevent aggregation. (Anat Ben-Zvi, Paolo De Los Rios, Giovanni Dietler, Pierre Goloubinoff, Active Solubilization and Refolding of Stable Protei Aggregates By Cooperative Unfolding Action of Individual Hsp70 Chaperones, The Journal of Biological Chemistry, Vol. 279, No. 36, Issue of September 3, 37298-37303, 2004). It is also reported that a Congo Red conjugate which binds to both FK506 binding protein and beta-amyloid peptide hinders the amyloid fibrillation. (Science, 306, 865, 2004)
In case of insulin, there are many methods to minimize insulin fibrillation that leads to aggregate precipitation from solution. Addition of zinc induces insulin to form a hexamer complex which contains two zinc per six insulins, and this complex is much more stable than monomeric insulin. Phenol, m-cresol are also used to improve insulin stability, Svend Havelund of Novo Nordisk tried to improve stability of liquid insulin for pulmonary delivery by optimizing the additive ratios, achieving marginal improvement. (U.S. Pat. Nos. 6,211,144, and 6,489,292) Peter Boderke of Aventis Pharama used zinc, Tween-20, Tween-80, Poloxamer 171 to achieve insulin stability 5-7 folds longer before fibrils precipitate (U.S. Pat. No. 6,960,561 B2)
Monomeric insulin has found its application in formulations for pulmonary delivery where it showed its superiority over hexameric zinc insulin. Solomon Steiner et al of Mannkind Corporation formulated zinc free insulin into microspheres with a diketopiperazine bulk reagent (U.S. Pat. No. 6,652,885 B2) for inhalation, and Igor Conda et al of Aradigm corporation formulated insulin lispro into solution for nebulized inhalation. Those monomeric insulins showed their advantage primarily due to their superior solubility and faster absorption through lung membrane.
Chemical modification using polyethylene glycol is also used to improve protein stability. However, it changes the protein chemically, and often decreases the biological activity of the protein.